The standard method for searching for new chemical compounds which can effectively modulate biological processes employs the screening of pre-existing compounds in assays which have been designed to test particular properties of the compound being screened. Similarly, in designing compounds having desired physiochemical properties for general chemical applications, numerous compounds must be individually prepared and tested.
To reduce the time and expense involved in preparing and screening a large number of compounds for biological activity or for desirable physiochemical properties, technology has been developed for providing libraries of compounds for the discovery of lead compounds. Current methods for generating large numbers of molecularly diverse compounds focus on the use of solid phase synthesis. The generation of combinatorial libraries of chemical compounds by employing solid phase synthesis is well known in the art. For example, Geysen, et al. (Proc. Natl. Acac. Sci. USA, 3998 (1984) describe the construction of multi-amino acid peptide libraries; Houghton, et al. (Nature, 354, 84 (1991) and PCT Patent Pub. No. WO 92/09300) describe the generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery; Lam, et al. (Nature, 354, 82 (1991) and PCT Patent Pub. No. WO 92/00091) describe a method of synthesis of linear peptides on a solid support such as polystyrene or polyacrylamide resin.
The growing importance of combinatorial chemistry as an integral component of the drug discovery process has spurred extensive technological and synthetic advances in the field (Thompson, L. A.; Ellman, J. A. (1996) Chem. Rev. 96, 555-600). Founded in peptide synthesis devised by Merrifield, solid phase chemistry has emerged as the preeminent method for construction of small molecule combinatorial libraries (see e.g. Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154; (a) Terrett, N. K.; Gardner, M.; Gordon, D. W.; Kobylecki, R. J.; Steele, J. (1995) Tetrahedron 51(30), 8135-8173. (b) Gordon, E. M.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gallop, M. A. (1994) J. Med. Chem. 37, 1385-1401.).
It is known that a wide variety of organic reactions can be carried out on substrates immobilized on resins. These include, in addition to peptide synthesis, nucleophilic displacements on benzylic halides, halogenation, nitration, sulfonation, oxidation, hydrolysis, acid chloride formation, Friedel-Crafts reactions, reductions, metallation, and the like which are well known in the art. (See for example, Mathur, et al., "Polymers as Aids in Organic Chemistry", Academic Press, New York, 18 (1980) and Farrall, et al., J. Org. Chem., 41, 3877 (1976); "Solid Phase Organic Chemistry (SPOC)" and "Solid Phase Inorganic Chemistry (SPIC)", Chiron Mimotopes, pp. 1-31 (August 1995).
One variant on the use of substrates imobilized on resin beads employs polystyrene pins as supports for the substrates (see Geysen, et al., J. Immunol. Meth., 102, 259 (1987) and another variant employs a flat solid support in the form of a tape or a streamer (see PCT Patent Publication WO 96/16078, published May 30, 1996).
Although combinatorial chemistry may be attempted by employing traditional synthetic chemistry in the solution phase, this is essentially impractical due to the difficulty in separating complex mixtures of intermediates and final compounds from reagents and solvents. Traditional solution phase chemistry has been criticized as being unsuitable as a technique which aims to simultaneously produce a multiplicity of new products, since this would not allow physical separation among the different products. The products are therefore likely to be contaminated with excess reagents, by-products, etc., leading to difficulties in separation and purification.
Central to the power of solid phase synthesis is the ease by which reagents and solvents are removed simply by washing. This allows for the purification of resin-bound mixtures of great complexity, and the use of large reagent excesses to drive reactions to completion. The "infinite dilution" obtained on solid supports can also prevent side reactions which may occur in solution. Despite its advantages, nontrivial liabilities are associated with solid phase synthesis. Most notable is the often arduous task of modifying solution phase chemistry to the solid phase, with its potential pitfalls such as poor solvation, differential site accessibility, and incompatibility of the polymer support with reagents or reaction conditions. Often the most time-consuming aspect of combinatorial library synthesis is not construction of the library itself, but rather translation of solution phase chemistry to the solid phase. Although a significant portion of organic chemistry can be adapted to synthesis on the solid phase, some reactions, such as heterogeneous catalysis, would be exceedingly difficult to conduct on reagents linked to a solid phase support. In addition to synthetic complications which may arise from employing a solid support, few analytical techniques exist for characterization of resin-bound compounds. Even if NMR or IR analytical methods are attempted, they are impractical because the resultant spectra are generally of such low quality. Thus, it is very difficult to monitor reactions conducted on solid phase. Even analysis of cleaved intermediates can be ambiguous, since the harsh cleavage conditions that are often required may be detrimental to the molecules of interest.
Accordingly, alternative methodology for the preparation of combinatorial libraries which overcomes the drawbacks of solid phase synthesis would provide a significant advance in the field.
Classical solution phase techniques have been developed in an attempt to overcome the drawbacks of solid phase synthesis. One approach is to separate acidic and basic reagents from the resultant products by adding a water solution of additional acids or bases (See Boger, et al, J. Am. Chem. Soc., (Feb. 28, 1996)). A variant of this method takes advantage of extraction of the reagents or products into a perfluorocarbon solvent.
Another approach, termed liquid-phase combinatorial synthesis (LPCS), in which combinatorial libraries are synthesized on soluble polyethylene glycol (PEG) supports has been recently described (Han, H., Wolfe, M. M., Brenner, S., Janda, K. D. (1995) Proc. Natl. Acad. Sci. USA 92, 6419-6423; EPO Patent Publication No. EP 0,772,623 (May 14, 1997)). In this method, monofunctional PEG which falls within a certain molecular weight range is used as a support for synthetic reactions. When the reactions are complete, ether is added to the solution which causes precipitation of the PEG, which is then isolated by filtration. This precipitation/crystallization of the PEG-protected molecules from ether allows for removal of reagents and solvents by filtration, thus combining the advantages of solution phase chemistry and the utility of solid phase purification. This technology has been reviewed in Science, 272, 1266-1268 (May 31, 1996).
The use of a soluble polyvalent support in accordance with the present invention provides significant advantages with respect to solid phase synthesis of combinatorial libraries, including: (1) solution phase synthesis obviates the need to modify chemistry to the solid phase; (2) intermediates may be routinely characterized by a variety of analytical methods, including .sup.1 H and .sup.13 C NMR, IR, UV and mass spectrometry, with the generation of spectra of generally high resolution through NMR being made possible; (3) because multiple copies of each molecule are synthesized per polyvalent support, extremely high loadings may be attained; (4) size-based purification is general, since it does not rely on other physical differences between support-bound compounds and reagents; the use of large reagent excesses are also permitted; and (5) polyvalent supports offer a flexible framework that may be engineered to exhibit properties necessary for their desired applications. Accordingly, combinatorial chemistry on soluble polyvalent supports provides a valuable alternative to solid phase synthesis.